The long-term goal of this project is to identify and characterize novel proteins that regulate mitochondrial heme metabolism, particularly proteins that play a role in mitochondrial heme/porphyrin transport. This project has implications for identifying genetic modifiers for erythroid porphyria and anemia, and thus has public health significance. Our lab has identified Tmem14c as a novel mitochondrial protein that is required for terminal erythroid differentiation and hemoglobinization in vertebrates. Heme synthesis enzymes are present in Tmem14c-deficient murine erythroleukemia (MEL) cells, and preliminary data from our lab indicate that iron transport in Tmem14c-deficient MEL cells is comparable to wild-type MEL cells. However, Tmem14c-deficient MEL cells synthesize decreased levels of heme and protoporphyrin IX. These data suggest that Tmem14c may facilitate mitochondrial porphyrin transport, thereby playing a key role in heme/porphyrin metabolism. In Aim 1, we will test this hypothesis by identifying the block in porphyrin metabolism in Tmem14c deficient cells, comparing the levels of uroporphyrinogen III (cytoplasmic), coproporphyrinogen III (mitochondrial/cytoplasmic) and protoporphyrin IX (mitochondrial) in the mitochondrial and cytoplasmic fractions of wild-type and Tmem14c-deficient MEL cells. In this manner, we will determine if the block in heme synthesis in Tmem14c-deficient cells is a result of defective porphyrin trafficking. As Tmem14c forms higher-order complexes with other proteins, we hypothesize that Tmem14c's interactions with its partners play a crucial role in regulating its function. In Aim 2, we will identify Tmem14c partner and characterize their role in regulating Tmem14c in the heme synthetic pathway; we will identify interacting proteins that are required for terminal erythroid differentiation, heme synthesis and porphyrin transport, and focus on characterizing proteins that regulate Tmem14c's localization and protein stability. Collectively, our studies will contribute to our lon-term objective of understanding regulatory mechanisms controlling heme metabolism, and the interaction of heme metabolism with erythropoiesis. The specific aims in this proposal are of particular significance because studies of Tmem14c function will shed light on the poorly understood porphyrin trafficking pathways that are central to the regulation of heme metabolism and erythropoiesis. These experimental aims are a logical continuation of my graduate work in hematopoiesis, but provide a framework with which to obtain substantial training in the use of the zebrafish as an animal model and additional techniques that will prove invaluable for further studies in erythroid biology.